Biomedical subjects

David M Bader

Publications and source records attributed to David M Bader.

13 recordsLinked to original sources

PubMed2006

CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25.

SNAP-25 is a component of the SNARE complex that is involved in membrane docking and fusion. Using a yeast two-hybrid screen, we identify a novel interaction between SNAP-25 and cytoplasmic Lek1 (cytLEK1), a protein previously demonstrated to associate with the microtubule network. The binding domains within each protein were defined by yeast two-hybrid, coimmunoprecipitation, and colocalization studies. Confocal analyses reveal a high degree of colocalization between the proteins. In addition, the endogenous proteins can be isolated as a complex by immunoprecipitation. Further analyses demonstrate that cytLEK1 and SNAP-25 colocalize and coprecipitate with Rab11a, myosin Vb, VAMP2, and syntaxin 4, components of the plasma membrane recycling pathway. Overexpression of the SNAP-25-binding domain of cytLEK1, and depletion of endogenous Lek1 alters transferrin trafficking, consistent with a function in vesicle recycling. Taken together, our studies indicate that cytLEK1 is a link between recycling vesicles and the microtubule network through its association with SNAP-25. This interaction may play a key role in the regulation of the recycling endosome pathway.

Animals↗

PubMed2006

Bves, a member of the Popeye domain-containing gene family.

Bves was discovered through subtractive screens designed to identify heart-enriched transcripts. Bves is a transmembrane protein that possesses a highly conserved structure among species of the animal kingdom. Various approaches have been used to elucidate the expression pattern of Bves mRNA and protein as well as its function in developing and mature organisms. Emerging evidence indicates that this protein is present in muscle and epithelia of developing embryos and the adult. In vitro functional studies predict a role in cell-cell interaction and/or adhesion. In vivo analysis of protein function is very limited at present, but recent work in Xenopus supports the importance of Bves in epithelial integrity. Presented in this review is a compilation of published findings concerning Bves gene and protein characteristics, expression patterns in embryos and cells, and functional significance as determined thus far. Presently, the literature supports a hypothesis that Bves is essential to the junctional architecture of muscle and epithelial cell types. Although there remain aspects of Bves structure, expression, and function that are not completely resolved, now is an appropriate time to summarize current knowledge about this protein, the remaining questions, and what its potential role in development might be. This review will serve as a departure point for others who become interested in the study of this highly conserved protein.

Amino Acid Sequence↗

PubMed2006

Characterization of Bves expression during mouse development using newly generated immunoreagents.

Bves (blood vessel/epicardial substance) is a transmembrane protein postulated to play a role in cell-cell interaction/adhesion. It was independently isolated by two groups as a gene product highly enriched in the developing heart. Disagreement exists about its expression during development. Most notably, the expression of Bves in non-muscle cells is disputed. Determining the expression profile of Bves is a critical initial step preceding the characterization of protein function in development and in the adult. We have generated new monoclonal antibodies against mouse Bves and used these immunoreagents to elucidate Bves expression in development. As expected, we detect Bves in myocytes of the developing heart throughout development. In addition, skeletal and smooth muscle cells including those of the coronary system express Bves. Finally, specific, but not all, epithelial derivatives of the three germ layers are stained positively with these monoclonal antibodies. Protein expression in cultured epithelial and muscle cell lines corroborate our in vivo findings. Taken together, these results demonstrate the expression of Bves in a wide range of epithelial and muscle cells during mouse embryogenesis and indicate a broad function for this protein in development, and show that these newly generated reagents will be invaluable in further investigation of Bves.

Amino Acid Sequence↗

PubMed2006

Pancreatic islet production of vascular endothelial growth factor--a is essential for islet vascularization, revascularization, and function.

To investigate molecular mechanisms controlling islet vascularization and revascularization after transplantation, we examined pancreatic expression of three families of angiogenic factors and their receptors in differentiating endocrine cells and adult islets. Using intravital lectin labeling, we demonstrated that development of islet microvasculature and establishment of islet blood flow occur concomitantly with islet morphogenesis. Our genetic data indicate that vascular endothelial growth factor (VEGF)-A is a major regulator of islet vascularization and revascularization of transplanted islets. In spite of normal pancreatic insulin content and beta-cell mass, mice with beta-cell-reduced VEGF-A expression had impaired glucose-stimulated insulin secretion. By vascular or diffusion delivery of beta-cell secretagogues to islets, we showed that reduced insulin output is not a result of beta-cell dysfunction but rather caused by vascular alterations in islets. Taken together, our data indicate that the microvasculature plays an integral role in islet function. Factors modulating VEGF-A expression may influence islet vascularity and, consequently, the amount of insulin delivered into the systemic circulation.

Animals↗

PubMed2005

Bves modulates epithelial integrity through an interaction at the tight junction.

We first identified Bves (blood vessel/epicardial substance) as a transmembrane protein that localized to the lateral compartment of the epithelial epicardium. Bves traffics to sites of cell-cell contact in cultured epicardial cells and promotes adhesion following transfection into non-adherent fibroblastic L-cells, reminiscent of a cell adhesion molecule. Currently, no function for Bves in relation to epithelial cell adhesion has been identified. We hypothesize that Bves plays a role at cell junctions to establish and/or modulate cell adhesion or cell-cell interactions in epithelial cell types. In this study, we demonstrate that Bves regulates epithelial integrity and that this function may be associated with a role at the tight junction (TJ). We report that Bves localizes with ZO-1 and occludin, markers of the TJ, in polarized epithelial cell lines and in vivo. We find that the behavior of Bves following low Ca2+ challenge or TPA treatment mimics that observed for ZO-1 and is distinct from adherens junction proteins such as E-cadherin. Furthermore, GST pull-down experiments show an interaction between ZO-1 and the intracellular C-terminal tail of Bves. Finally, we demonstrate that Bves modulates tight junction integrity, as indicated by the loss of transepithelial resistance and junction protein localization at the membrane following Bves knock-down in cultured cells. This study is the first to identify a function for Bves in epithelia and supports the hypothesis that Bves contributes to establishment and/or maintenance of epithelial cell integrity.

Animals↗

PubMed2005

Cytoplasmic LEK1 is a regulator of microtubule function through its interaction with the LIS1 pathway.

LIS1 and nuclear distribution gene E (NudE) are partner proteins in a conserved pathway regulating the function of dynein and microtubules. Here, we present data that cytoplasmic LEK1 (cytLEK1), a large protein containing a spectrin repeat and multiple leucine zippers, is a component of this pathway through its direct interaction with NudE, as determined by a yeast two-hybrid screen. We identified the binding domains in each molecule, and coimmunoprecipitation and colocalization studies confirmed the specificity of the interaction between cytLEK1 and NudE. Confocal deconvolution analysis revealed that cytLEK1 exhibits colocalization with endogenous NudE and with the known NudE binding partners, LIS1 and dynein. By localizing the NudE-binding domain of cytLEK1 to a small domain within the molecule, we were able to disrupt cytLEK1 function by using a dominant negative approach in addition to LEK1 knockdown and, thus, examine the role of the cytLEK1-NudE interaction in cells. Consistent with a defect in the LIS1 pathway, disruption of cytLEK1 function resulted in alteration of microtubule organization and cellular shape. The microtubule network of cells became tightly focused around the nucleus and resulted in a rounded cell shape. Additionally, cells exhibited a severe inability to repolymerize their microtubule networks after nocodazole challenge. Taken together, our studies revealed that cytLEK1 is essential for cellular functions regulated by the LIS1 pathway.

Animals↗

PubMed2005

The serosal mesothelium is a major source of smooth muscle cells of the gut vasculature.

Most internal organs are situated in a coelomic cavity and are covered by a mesothelium. During heart development, epicardial cells (a mesothelium) move to and over the heart, undergo epithelial-mesenchymal transition (EMT), and subsequently differentiate into endothelial and vascular smooth muscle cells. This is thought to be a unique process in blood vessel formation. Still, structural and developmental similarities between the heart and gut led us to test the hypothesis that a conserved or related mechanism may regulate blood vessel development to the gut, which, similar to the heart, is housed in a coelomic cavity. By using a combination of molecular genetics, vital dye fate mapping, organ culture and immunohistochemistry, we demonstrate that the serosal mesothelium is the major source of vasculogenic cells in developing mouse gut. Our studies show that the gut is initially devoid of a mesothelium but that serosal mesothelial cells expressing the Wilm's tumor protein (Wt1) move to and over the gut. Subsequently, a subset of these cells undergoes EMT and migrates throughout the gut. Using Wt1-Cre genetic lineage marking of serosal cells and their progeny, we demonstrate that these cells differentiate to smooth muscle of all major blood vessels in the mesenteries and gut. Our data reveal a conserved mechanism in blood vessel formation to coelomic organs, and have major implications for our understanding of vertebrate organogenesis and vascular deficiencies of the gut.

Animals↗

PubMed2004

Bves expression during avian embryogenesis.

Bves (blood vessel/epicardial substance) is a transmembrane protein postulated to play a role in cell adhesion. While it is clear that Bves and gene products of the same family are expressed in adult striated muscle cells, the distribution of these proteins during development has not been critically examined. An understanding of the expression pattern of Bves is essential for a determination of protein function and its role in embryogenesis. In this study, we present an expression analysis of Bves during chick gastrulation and germ layer formation. Our data show that Bves is expressed in epithelia of all three germ layers early in development. Furthermore, Bves protein is observed in epithelial tissues during organogenesis, specifically the developing epidermis, the gut endoderm, and the epicardium of the heart. These data support the hypothesis that Bves may play a role in cell adhesion and movement of epithelia during early embryogenesis.

Amino Acid Sequence↗

PubMed2004

Bves is expressed in the epithelial components of the retina, lens, and cornea.

PURPOSE: To demonstrate the expression pattern and subcellular localization of Bves/Pop1a protein, a newly identified cell adhesion molecule, during eye development and corneal regeneration. METHODS: Staged embryonic and adult eyes were assayed using fluorescence immunohistochemistry to detect the Bves protein. A human corneal epithelial (HCE) cell line was used as a model to examine Bves localization during corneal growth and regeneration, with and without antisense morpholino treatment. RESULTS: The data detail the expression and localization of Bves protein before, during, and after differentiation of the eye. In these analyses, Bves was localized to an apical-lateral position in the initial epithelial primordia of the eye. Later, Bves became localized to specific cell types and subcellular domains in the retina, lens, and cornea, indicating changes in Bves expression in the differentiated eye. Finally, an in vitro model of corneal wound healing showed that Bves staining was missing at the epithelial surface during cellular migration across the wound, but it reappeared at points of cell contact during the reinitiation of epithelial continuity. When epithelial sheets were treated with Bves antisense morpholinos to inhibit Bves function, disruption of epithelial integrity was observed. After injury, similar treatment resulted in an acceleration of cell movement at the wound surface but regeneration of an intact epithelium was ultimately impeded. CONCLUSIONS: Taken together, these studies suggest that Bves is expressed in epithelial elements of the developing eye and may have a role in corneal epithelial growth and regeneration.

Animals↗

PubMed2003

Membrane topology of Bves/Pop1A, a cell adhesion molecule that displays dynamic changes in cellular distribution during development.

We investigated the membrane topology of Bves/Pop1A as a foundation to dissect the molecular basis and function of Bves/Pop1A trafficking during development. Bves contains two asparagine-linked glycosylation sites within the amino terminus and three putative membrane domains. Therefore, glycosylation assays were performed to determine if the amino terminus of Bves is delivered into the endoplasmic reticulum lumen and glycosylated. We establish that Bves from chick heart and transfected cells is glycosylated, implying that the amino terminus of cell surface molecules is extracellular. Three biochemically distinct approaches were utilized to determine the orientation of the carboxyl terminus of Bves. First, glycosylation of Bves at exogenous sites within the carboxyl terminus was only observed in a construct that lacked the third membrane domain, which presumably reversed the orientation of the carboxyl terminus. Second, co-expression of full-length Bves with soluble, carboxyl-terminal Bves constructs that reside in different subcellular compartments revealed that Bves-Bves interactions occur in the cytoplasm. Third, the immunoreactivity of endogenous Bves at the cell surface of epicardial cells was dramatically enhanced with detergent. These results suggest that the membrane topology of cell surface Bves/Pop1A is composed of an extracellular amino terminus, three transmembrane domains, and a cytoplasmic carboxyl terminus. We therefore hypothesize that the carboxyl terminus regulates the cellular distribution of Bves/Pop1A during coronary vessel development.

3T3 Cells↗

PubMed2003

Epicardial/Mesothelial cell line retains vasculogenic potential of embryonic epicardium.

Recent work has demonstrated the importance of the epicardium in the development of the heart. During embryogenesis, these epithelial cells provide the progenitors for the epicardium, coronary smooth muscle, endothelium, and cardiac fibroblasts. The epicardium sends important signals to the developing myocardium. Still, analysis of these epithelial cells has lagged behind that of other cardiac cell types largely because of the lack of a defined experimental cell system in which epicardial cell differentiation can be studied. The present report examines the developmental potential of a cell line derived from rat epicardial mesothelial cells. These analyses demonstrate that the cell line retains many characteristics of the intact epithelium, including the ability to form a polarized epithelium and express many epicardial genes. Our data show for the first time that these cells retain the ability to produce mesenchyme in response to specific growth factors and, importantly, to generate smooth muscle cells. Thus, this study provides evidence that these cells can serve as an important model system for the analysis of the cellular and molecular mechanisms that govern epicardial development and function.

Animals↗

PubMed2002

Development of the coronary vessel system.

Formation of the coronary vessels is a fundamental event in heart development. Congenital abnormalities in the coronary system can have major deleterious effects on heart function. It is also possible that subtle variation in the patterning of coronary vessels has significant but uncharacterized effects on myocardial structure and function. In addition, generation of the coronary vascular system represents a complex system for analysis of regulation of cell fate determination, cell and epithelial migration, epithelial/mesenchymal transition, and patterning of a complex three-dimensional structure. In this review, we present the descriptive embryology of this process as well as the recent data that shed light on the unique developmental mechanisms underlying generation of coronary vessels. This review also attempts to identify areas where additional research is needed and highlights the questions that must be answered for a meaningful understanding of coronary vessel development.

Animals↗

PubMed2002

Hole is a novel gene product expressed in the developing heart and brain.

Hole is a novel gene product isolated from a chick heart subtractive hybridization. Hole is a six-transmembrane protein (predicted size 311 and 317 amino acids in chick and mouse) expressed in the cardiac crescent and later in the myocardium of the developing chick heart, as well as in the fusing neural tube and ganglia. Mouse hole is not expressed in the developing heart, although it does share neural expression seen in the chick.

Amino Acid Sequence↗